The invention relates generally to assays for determining cysteine concentration and cysteine synthase activity.
Cysteine synthase (EC 4.2.99.8) is an enzyme involved in the final step of cysteine biosynthesis in a number of prokaryotic and eukaryotic organisms and catalyzes the formation of the amino acid L-cysteine from O-acetyl-L-serine (OAS) and inorganic sulfide. Inorganic sulfide is fixed by the activity of cysteine synthase. The co-factor pyridoxal phosphate converts serine to OAS, which is then converted by cysteine synthase can convert the OAS to cysteine (Saito, K., Kurosawa, M, and Murakoshi, I.).
Cysteine synthase genes have been characterized from a number of plants, and in most cases consist of a small multi-gene families, with differing cellular localization for each family member (Nakamura, T., Yamaguchi, Y., and Sano, H.). At least ten different cysteine synthase genes have been identified in Arabidopsis thaliana. The Arabidopsis cysteine synthase gene, AtcysC1, encodes a mitochondrial protein having both cysteine synthase activity (EC 4.2.99.8) and xcex2-cyanoalanine synthase activity (EC 4.4.1.9). Yamaguchi et al. (2000) Plant Cell Physiol 41:465-476.
Commonly assigned co-pending U.S. patent application Ser. No. 09/697,225, filed Oct. 26, 2000, discloses that cysteine synthase activity is essential for plant growth and development, and can therefore be used as a herbicide target. growth and development, and can therefore be used as a herbicide target. Accordingly, it would be desirable to screen herbicide candidates for inhibition of cysteine synthase activity in high throughput assays.
Current or existing assays for cysteine synthase activity include: 1) the measurement of cysteine as a red ninhydrin complex (Gaitonde (1967) Biochem J. 104:627-633); 2) derivatization of the thiol group of cysteine and identification by HPLC methods (Fahey et al. (1987) Methods in Enzymology 143:85-96; Cooper et al. (1987) Methods in Enzymology 143:141-143); 3) determination of pyruvic acid after reacting cysteine with cysteine desulfhydrase (Wedding (1987) Methods in Enzymology 143:29-31); and by protein binding assays (Smith et al. (1987) Methods in Enzymology 143:144-148). However, none of these methods are readily adaptable to high throughput screening assays.
The present invention provides assays for determining cysteine concentration and cysteine synthase activity, and methods for identifying herbicides, fungicides and bactericides that function by inhibiting cysteine synthase activity.
Cysteine concentration can be quantitated by contacting cysteine with a coumarin dye capable of conjugating with cysteine but not conjugating to a significant extent with O-acetyl serine or sulfide to produce a cysteine-coumarin conjugate; exciting the conjugate with UV light; and detecting fluorescent light emitted by the conjugate. Preferred coumarin dyes are CPM and DACM Cysteine synthase activity can be determined by combining O-acetyl-L-serine, sulfide and cysteine synthase to form a reaction mixture under conditions suitable for the cysteine production; contacting the reaction mixture with an appropriate coumarin dye, for example, CPM and/or DACM; subjecting the reaction mixture to UV light; and detecting fluorescent light emission. The amount of cysteine produced can be determined by the amount of fluorescence, and the activity of the cysteine synthase determined by the amount of cysteine produced.
Methods for identifying test compounds that function as herbicides, fungicides or bactericides involve combining O-acetyl-L-serine, sulfide and a suitable plant, fungal or bacterial cysteine synthase to form a reaction mixture in the presence and absence of the test compound; contacting the reaction mixture with an appropriate coumarin dye; subjecting the reaction mixture to UV light; and detecting fluorescent light emission. The amount of the fluorescent light emission in the presence and absence of the test compound is compared. A decrease in the amount of the fluorescent light emission in the presence of the test compound indicates that the test compound is a herbicidal, fungicidal or bactericidal candidate.